Human umbilical cord blood stem cells differentiate into nasal ciliated epithelial cells

doi:10.3969/j.issn.2095-4344.2016.32.008

Abstract

Abstract:

BACKGROUND: Damage to nasal ciliated epithelial cells can lead to a severe injury in nasal biological function. Compared with other adult stem cells, human umbilical cord blood stem cells have better differentiation potential.
OBJECTIVE: To explore the feasibility of human umbilical cord blood stem cells differentiating into nasal ciliated epithelial cells through in vitro culture and induction techniques.
METHODS: Normal and healthy umbilical cord blood samples were collected to isolate human umbilical cord blood stem cells, followed by identification and subculture in vitro. Umbilical cord blood stem cells at passage 3 were infected with recombinant adeno-associated virus carrying enhanced green fluorescent protein and cultured using air liquid interface culture method. Thereafter, PCR assay was employed for detecting MUCS expression in cultured stem cells at 1 and 2 weeks after induction, and immunofluorescent staining for FOXJ1 was performed at 3 weeks.
RESULTS AND CONCLUSION: After subculture, passage 3 umbilical cord blood stem cells that could express stem cell surface markers were visible in a uniform shape and had good refraction. After 3 hours of gene transfection, green fluorescence issued from the passage 3 cells were visible, and the cell positive rate was up to 96.2% until 48 hours, indicating good transfection efficiency. RT-PCR findings showed that MUC8 mRNA had no expression in the umbilical cord blood stem cells, but expressed strongly in the nasal ciliated epithelial cells, whose expression was weak at 1 week of culture and increased at 2 weeks. Additionally, the positive expression of FOXJ1 red fluorescence was observed under the transfection of green fluorescent protein. These results suggest that human umbilical cord blood stem cells could differentiate into nasal epithelial cells under suitable conditions.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Tissue Engineering, inusitis

CLC Number:

R394.2

Dong Jin-hui, Ren Xiu-min, Xu Ou, Wang Jian-xing. Human umbilical cord blood stem cells differentiate into nasal ciliated epithelial cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(32): 4764-4770.

Figures/Tables 1

2.1 人脐血干细胞显微形态原代人脐血干细胞接种后，于37 ℃，体积分数5%CO2饱和湿度培养箱培养1周后，干细胞正常贴壁生长，出现多种形态，主要形态呈现为圆形细胞、梭形或不规则，有形成集落的趋势。换取培养基后继续于培养箱中培养至第4周时，脐血干细胞融合达80%以上，可正常传代。干细胞传至第3代时，光学显微镜下观察，可见脐血干细胞形态较均一，折光性良好，见图1。 2.2 人脐血干细胞表面抗原标记物表达特征经流式细胞仪检测结果显示，P3代脐血间充质干细胞稳定地高表达CD13和CD44等表面抗原标记物，阳性率分别为86.18%和90.30%，弱表达CD34和CD45等造血细胞标志，表明间充质干细胞是存在于脐血中区别于造血细胞的一群非定向干/祖细胞，这与骨髓间充质干细胞的表面抗原标志相一致，见图2。 2.3 人脐血干细胞的转染及转染率结果第3代人脐血干细胞被重组腺相关病毒转染3 h后，于荧光倒置显微镜下观察，可见第3代干细胞呈现出绿色荧光(图3)，而对照组细胞未见绿色荧光。干细胞培养48 h后，经流式细胞仪检测，细胞阳性率达96.2%，说明干细胞转染效果较好。 2.4 MUC8基因的表达 RT-PCR结果显示：人脐血干细胞MUC8 mRNA无表达；鼻黏膜上皮细胞MUC8 mRNA呈现出强表达，而且随重组腺相关病毒诱导培养时间的延长，培养1周时MUC8 mRNA呈现弱表达，培养2周后有一定的增强。内参照基因为GAPDH，阳性条带为308 bp，见图4。 2.5 细胞免疫荧光染色检测结果人脐血干细胞培养3周后进行免疫荧光染色，显微镜下未检测到β-Tubulin表达，而在转染的绿色荧光蛋白背景下可观测到FOXJ1红色荧光呈现阳性表达结果。共聚焦显微镜下可观察到部分标记绿色荧光的脐血干细胞细胞核内可见红色 FOXJ1荧光表达，见图5。"

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